Huperzine A and analogs thereof

ABSTRACT

The present invention relates to a method for the synthesis of certain bridged fused ring pyridines. Such bridged fused ring pyridines can be converted to huperzine A and analogs of huperzine A. The present invention also covers such bridged fused ring pyridines, compounds utilized for the preparation of the bridged fused ring pyridines and analogs of huperzine A.

This is a continuation, of application Ser. No. 08/279,372, filed Jul.22, 1994now U.S. Pat. No. 5,663,344, which is a continuation ofapplication Ser. No. 07/871,774, filed Apr. 21, 1992, now abandoned,which is a continuation of application Ser. No. 07/499,122, filed Mar.23, 1990, now issued U.S. Pat. No. 5,106,979, which is a division ofapplication Ser. No. 07/313,118, filed Feb. 21, 1989, now issued U.S.Pat. No. 4,929,731.

1. TECHNICAL FIELD

The present invention relates to a method for the synthesis of certainbridged fused ring pyridines. Such bridged fused ring pyridines can beconverted to huperzine A and analogs of huperzine A. The presentinvention also covers such bridged fused ring pyridines, compoundsutilized for the preparation of the bridged fused ring pyridines andanalogs of huperzine A.

2. BACKGROUND OF THE INVENTION

Huperzine A, which is a Lycopodium alkaloid, has been isolated from theplant Huperzia serrata. It has been shown to inhibit the cholinesteraseenzyme and, therefore, has been tested for the treatment of diseases ofthe cholinergic system. For example, Huperzine A is being studied forthe treatment of myasthenia gravis, Alzheimer's dementia and for theimprovement of senile memory loss. See J. Liu, et al., The Structures ofHuperzine A and B, Two New Alkaloids Exhibiting MarkedAnticholinesterase Activity, Can. J. Chem., 64, 837-839 (1986).

3. SUMMARY OF THE INVENTION

The present invention relates to a method for the synthesis of a bridgedfused ring pyridine of the general formula I: ##STR1## which comprises:(A) contacting a fused ring pyridine having general formula II: ##STR2##with an unsaturated carbon bridge having the general formula III:##STR3## in a suitable solvent comprising an amine base catalyst havinga pKa of from about 11 to about 20 to form the bridged fused ringpyridine of general formula I;

wherein:

R₁ ¹ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkoxy;

R₁ ² is selected from the group consisting of C₁ -C₈ linear or branchedalkyl;

R₂ ³ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₃ ¹ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkoxy;

R₃ ² is selected from the group consisting of C₁ -C₈ linear or branchedalkyl;

R₃ ³ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

n₁ is an integer from 0 to 4; and

n₂ is an integer from 0 to 4;

with the proviso:

R₁ ² =R₃ ² ; R₁ ¹ =R₃ ¹ ; R₂ ³ =R₃ ³ and n₁ =n₂.

The bridged fused ring pyridine of the general formula I can beconverted to the compound of general formula IV, which includeshuperzine A and the analogs of huperzine A of the present invention:##STR4## wherein: R₄ ³ is selected from the group consisting of H and C₁-C₈ linear or branched alkyl;

R₄ ⁴ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₄ ⁵ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₄ ⁶ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₄ ⁷ is selected from the group consisting of H and C₁ -C₈ linear ofbranched alkyl;

n₃ is an integer from 0 to 4;

p is 0 or 1;

- - - represents a double bond between carbon 14 and 15 or a double bondbetween carbon 8 and 15;

with the proviso: R₂ ³ =R₃ ³ =R₄ ³.

The compounds of general formula IV are capable of inhibiting thecholinesterase enzymes.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for the synthesis of a bridgedfused ring pyridine of general formula I: ##STR5## The method comprisescontacting a fused ring pyridine of the general formula II: ##STR6##with an unsaturated carbon bridge having the general formula III:##STR7## in suitable solvent comprising an amine base catalyst having apKa of from about 11 to about 20 to form the bridged fused ring pyridineof general formula I wherein:

R₁ ¹ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkoxy;

R₁ ² is selected from the group consisting of C₁ -C₈ linear or branchedalkyl;

R₂ ³ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₃ ¹ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkoxy;

R₃ ² is selected from the group consisting of C₁ -C₈ linear or branchedalkyl;

R₃ ³ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

n₁ is an integer from 0 to 4; and

n₂ is an integer from 0 to 4;

with the proviso:

R₁ ² =R₃ ² ; R₁ ¹ =R₃ ¹ ; R₂ ³ =R₃ ³ and n₁ =n₂.

In a preferred embodiment:

R₁ ¹ is OCH₃ ;

R₁ ² is CH₃ ;

R₂ ³ is CH₃ ;

R₃ ¹ is OCH₃ ;

R₃ ² is CH₃ ;

R₃ ³ is CH₃ ;

n₁ is 1; and

n₂ is 1. This embodiment is preferred because with such embodimenthuperzine A can be readily made.

It has now been discovered that the use of a suitable solvent comprisingan amine base catalyst having a pKa of from about 11 to about 20 readilypermits (>90% yield) the unsaturated carbon bridge of general formulaIII to be readily added to the fused ring pyridine of general formulaII. Without being bound by theory, it is believed that the unsaturatedcarbon bridge is added to the fused ring pyridine by Michael reactionfollowed by an aldol reaction.

It is believed that the use of the amine base catalyst having a pKa offrom about 11 to about 20, and preferably about 14, is what permits suchreaction to proceed so readily. (Such pKa is measured relative towater.) For example, the amine base catalyst triethylamine, whose pKa isabout 10, fails to permit the reaction to proceed. Also, the basecatalyst sodium methoxide, which is not an amine base catalyst, but hasa pKa of about 16, also does not permit the reaction to proceed.Preferred amine base catalysts are 1,1,3,3-tetramethylguanidine (TMG)and diazabicycloundecene, with TMG being most preferred.

It is believed that any suitable solvent that permits the reaction toproceed can be utilized, but a polar solvent is preferred and a polaraprotic solvent is even more preferred. Examples of polar aproticsolvents are methylene chloride, chloroform, dimethylformamide, dimethylsulfoxide, and tetrahydrofuran (THF), with methylene chloride beingpreferred.

It should be noted that no stereochemistry is implied by the generalformulas utilized in the present invention; all stereoisomers areincluded in each general formula.

4.1 PREPARATION OF FUSED RING PYRIDINE

The fused ring pyridine of general formula II can be prepared byutilizing SCHEME I, below.

The starting material is a monoprotected diketone 1. The monoprotecteddiketone 1 is reacted with pyrrolidine and the resulting enamine isheated with acrylamide to provide the lactam 2. The lactam intermediateis then converted by a dehydrogenation procedure to the pyridone 3.Next, this pyridone is alkylated on oxygen to provide the alkoxypyridinederivative R¹ =OR). The ketone carbonyl group is now deprotected and anα-carboalkoxylation reaction is carried out to provide the β-keto estermaterial 5, which corresponds to a molecule of general formula II.

Pyridone 3 can also be converted to a fused ring pyridine of generalformula II (or 5) with R¹ =H by reducing the pyridone carbonyl group of3 to hydroxyl and carrying out a subsequent dehydration step. Removal ofthe ketal protecting group and a carboalkoxylation reaction the provideII (or 5) with R¹ =H. ##STR8##

4.2. CONVERSION OF THE BRIDGED FUSED RING PYRIDINE TO HUPERZINE A ANDANALOGS OF HUPERZINE A

The bridged fused ring pyridine of general formula I can be converted toa compound of general formula IV, which includes huperzine A and theanalogs of huperzine A of the invention: ##STR9## wherein: R₄ ³ isselected from the group consisting of H and C₁ -C₈ linear or branchedalkyl;

R₄ ⁴ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₄ ⁵ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₄ ⁶ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₄ ⁷ is selected from the group consisting of H and C₁ -C₈ linear ofbranched alkyl;

n₃ is an integer from 0 to 4;

p is 0 or 1;

- - - represents a double bond between carbon 14 and 15 or a double bondbetween carbon 8 and 15;

with the proviso: R₂ ³ =R₃ ³ =R₄ ³.

Since huperzine A is the preferred compound to synthesize, preferably:

R₄ ³ is CH₃ ;

R₄ ⁴ is CH₃ ;

R₄ ⁵ is H;

R₄ ⁶ is H;

R₄ ⁷ is H;

n₃ is 1;

p is 0

- - - represents a double bond between carbon 8 and 15.

Even more preferred of such preferred embodiment is the E-stereoisomerof general formula IV, which represents huperzine A.

The compounds of general formula IV can be prepared by utilizing schemeII, hereinbelow.

The bridged fused ring pyridine 6 is converted to the bridged ketone 7by various dehydrating conditions. It is preferred that the alcohol isactivated for elimination by transformation to its mesylate derivative,which is then heated in sodium acetate and acetic acid to provide thebridged ketone 7.

The bridged ketone 7 is then reacted with the desiredalkylidenephosphorane (Ph₃ P═CHR⁴ wherein R⁴ is H or a C₁ -C₈ linear orbranched alkyl) in a suitable solvent, e.g. tetrahydrofuran or ether, toprovide the olefin 8 (note: where R⁴ is alkyl, a cis/trans mixture isformed). To obtain the olefinic product of predominantlyE-stereochemistry, a thermal isomerization reaction employing thiophenoland azoisobutyronitrile (AIBN) can be carried out to form ester 9.

The ester 9 is then transformed to the urethane derivative 10 bycarrying out a standard Curtius reaction, which comprises hydrolyzingthe ester to its acid, converting the acid to an acid chloride, followedby heating the acid chloride with sodium azide and then with methanol.

Urethane 10 can then be converted to amine 11 (huperzine A if R³ =R⁴=CH₃ and n=1) by effecting cleavage of both the alkyl group R¹ (where R¹=OR) and the carbomethoxy group by reacting urethane 10 with adealkylating agent, e.g. trimethylsilyl iodide.

In cases where R¹ in the accompanying structures is H, the pyridine ringis generated from the pyridone ring by a process involving pyridineN-oxide formation by use of a peracid, treatment with acetic anhydride,and then acid hydrolysis M. Katada, J. Pharm. Soc. Japan, 67, 51(1947)!. This conversion step is best performed prior to the TMSIpromoted cleavage of the urethane and can be performed at an earlierstage if required. Some modification may be required to avoid competingolefin epoxidation and/or oxidation of the acyclic amino group.

To procure the N-alkyl amino substituted derivatives 13 of huperzine A,the carbomethoxy group of 10 is removed by base hydrolysis, and theresulting free amine is sequentially alkylated to introduce R⁵ and R⁶ orR⁵ alone. An appropriate alkyl halide or tosylate, or a reductiveamination procedure is employed in introducing these groups. Lastly, thealkoxypyridine intermediate 12 (where R¹ =OR)is cleaved to the pyridone13 by O-dealkylation using a reagent such as TMSI.

Another aspect of the present invention is the pyridine intermediate 12having the general formula V: ##STR10## wherein: R₅ ¹ is selected fromthe group consisting of H and C₁ -C₈ linear or branched alkoxy;

R₅ ³ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₅ ⁴ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

R₅ ⁵ is selected from the group consisting of H and C1-C₈ linear orbranched alkyl;

R₅ ⁶ is selected from the group consisting of H and C₁ -C₈ linear orbranched alkyl;

n₄ is an integer from 0 to 4;

p is 0 or 1;

- - - represents a double bond between carbon 14 and 15 or a double bondbetween carbon 8 and 15.

Since huperzine A is the preferred compound to synthesize, preferably:

R₅ ¹ is OCH₃ ;

R₅ ³ is CH₃ ;

R₄ ⁴ is CH₃ ;

R₅ ⁵ is H;

R₅ ⁶ is H;

n₄ is 1;

p is 0; and

- - - represents a double bond between carbon 8 and 15.

Even more preferred of such preferred embodiment is the E-stereoisomerof general formula V, which is capable of being converted to huperzineA.

To obtain the huperzine analogs 14 containing an alkyl group (R⁷) on thepyridine ring nitrogen, intermediate 13 is deprotonated with a base andthe resulting anion is reacted with R⁷ X, where X is some suitableleaving group such as tosylate, mesylate, or halide.

To obtain the double bond regioisomer 16 of huperzine A, the double bondof 7 is subjected to an olefin isomerization reaction using a suitablemetal catalyst (e.g., Fe(CO)₅, (Ph₃ P)₄ Ru(MeCN), HCo(CO)₄), or byhydrating the double bond of 7 in the Markovnikov sense (H⁺, H₂ O), andthen carrying out a subsequent dehydration reaction.

The intermediate 15 is then used in the place of 7 in the foregoingreactions to provide the double bond regiosomers of 11, 13, and 14.

Saturated analogs 18 of huperzine A are readily obtained by subjecting 6to a Barton deoxygenation procedure the alcohol is converted to itsthiocarbonyl ester, and a tin hydride reduction carried out; see D. H.R. Barton and W. B. Motherwell, Pure Appl. Chem., 53, 15 (1981)!.Intermediate 17 is then carried through reaction steps identical tothose employed above for the conversion of 7 to 11, 13, or 14 in orderto acquire the saturated analogs 18 of huperzine A.

The one carbon homologs 20 of huperzine A can be obtained from 9 byreduction of ester to alcohol, conversion of alcohol to azide, andreduction of the azide to amine with LAH to afford 19. Thealkoxypyridine intermediate 19 (where R¹ =OR) is then transformed to 20by O-dealkylation using a reagent such as TMSI. Amine alkylationprocedures like those described for the conversion of 10 to 13 and 13 to14 can be employed to procure the analog 21. By starting with 15 andcarrying out a similar sequence of reactions to those describedhereinabove beginning with a Wittig step, access to the homolog 22 (adouble bond regioisomer of 21) can be achieved. ##STR11##

5. THE INHIBITION OF CHOLINESTERASE ENZYMES

The compounds of general formula IV are capable of inhibiting thecholinesterase enzymes and, therefore, are useful as pharmaceuticalagents for mammals, especially for humans, for the treatment ofdisorders wherein cholinesterase enzymes are involved. Examples of suchdisorders are myasthenia gravis, Alzheimer's dementia and theimprovement of senile memory loss.

The compounds of general formula IV can be administered to a humansubject either alone or, preferably, in combination withpharmaceutically acceptable carriers or diluents, optionally with knownadjuvants, such as alum, in a pharmaceutical composition, according tostandard pharmaceutical practice. The compounds can be administeredorally or parenterally, including intravenous, intramuscular,intraperitoneal, subcutaneous and topical administration.

For oral use of a compound of general formula IV, such compound can beadministered, for example, in the form of tablets or capsules, or as anaqueous solution or suspension. In the case of tablets for oral use,carriers which are commonly used include lactose and corn starch, andlubricating agents, such as magnesium stearate, are commonly added. Fororal administration in capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions are required for oral use,the active ingredient is combined with emulsifying and suspendingagents. If desired, certain sweetening and/or flavoring agents may beadded. For intramuscular, intraperitoneal, subcutaneous and intravenoususe, sterile solutions of the active ingredient are usually prepared,and the pH of the solutions should be suitably adjusted and buffered.For intravenous use, the total concentration of solutes should becontrolled in order to render the preparations isotonic.

When a compound according to general formula IV is used as in a humansubject, the daily dosage will normally be determined by the prescribingphysician with the dosage generally varying according to the age,weight, and response of the individual patient, as well as the severityof the patient's symptoms. However, in most instances, an effectivedaily dosage will be in the range of from about 0.05 mg/kg to about 1mg/kg of body weight, and preferably, of from 0.1 mg/kg to about 0.5mg/kg of body weight, administered in single or divided doses. In somecases, however, it may be necessary to use dosages outside these limits.

EXAMPLES

It should be noted that in the examples the numbers following the namedcompounds refer to the numbered compounds of Scheme I and Scheme II.Also, the variables stated in the examples correspond to the variablesof the general formulas.

6.1 Example I Synthesis of Huperzine A

Preparation of lactam 2 (n=1)

In a 500 mL round-bottomed flask equipped with a water separator and acondenser were placed 25 g (0.16 mol) of 1,4-cyclohexanedionemonoethylene ketal, 27 mL (0.32 mol) of pyrrolidine, 1 g ofp-toluenesulphonic acid, and 250 mL of benzene. The mixture was refluxeduntil no more water separated in the water separator. Benzene wasevaporated and the residue was dissolved in 250 mL of dioxane. To thissolution was added 34 g (0.48 mol) of acrylamide and the mixture wasrefluxed overnight. Water (100 mL) was added and the solution wasrefluxed for 12 h. After cooling down to room temperature, the dioxanewas removed by rotary evaporation and the aqueous residue was extractedwith CHCl₃. The extracts were washed with brine, dried with anhydrousMgSO₄, and filtered. After evaporation of the solvent, the residue waschromatographed on silica gel with ethyl acetate as the eluent. Theyield was 20 g (59%).

Preparation of Pyridine 3

(1.) N- Benzylation of 2 (n=1)

Potassium hydride (1.38 g, 0.0348 mol) was added in several portions toa mixture of the lactams 2 and 3 (4.85 g, 0.023 mol), benzyl chloride(5.3 mL, 0.0464 mol), and a catalytic amount of tetrabutylammoniumiodide in 250 ml of dry THF. The mixture was stirred at room temperatureovernight with the protection of a drying tube. Water was added dropwiseto quench the excess KH, and the THF was removed by rotary evaporation.The aqueous residue was extracted with ethyl acetate. The extracts werewashed with brine, dried with anhydrous MgSO₄, and filtered. Evaporationof the solvent, and purification of the residue by flash chromatography(ethyl acetate gave 6.95 g of the N-benzylated product (quantitativeyield).

(2.) Dehydrogenation of the N-benzylated product

To a solution of diisopropylamine (6.2 mL, 0.044 mol) in 100 mL of dryTHF at 0° C. under N₂ was added n-BuLi (24 mL of 1.6M n-BuLi in hexanes,0.038 mol). The solution was stirred at 0° C. for 20 min. and thencooled to -78° C. A solution of the above benzyl protected lactams (3.80g, 0.0127 mol) in 100 mL of dry THF was added at -78° C. The color ofthe reaction mixture immediately turned deep blue. After stirring at-78° C. for 2 h, a solution of phenylselenyl chloride (4.87 g, 0.0254mol) in 20 mL of dry THF was added dropwise, and the resulting solutionwas stirred at -78° C. for 15 min.

The solution was quenched with methanol (20 mL) and allowed to warm toroom temperature. The solution was then poured into a mixture of NaIO₄(10.88 g, 0.051 mol) in 300 mL of H₂ O-MeOH-THF (1:1:1). Another 100 mLof THF was used to rinse the reaction flask and combined with the abovemixture. The mixture was stirred at room temperature for 24 h.

THF and methanol were removed by rotary evaporation, and the aqueousresidue was extracted with ethyl acetate. Concentration of the ethylacetate solution gave a red syrup. The syrup was dissolved in 100 mL ofMeOH. Et₃ N (1.8 mL, 0.0127 mol) was added, and the solution wasrefluxed overnight. Concentration and column chromatography (ethylacetate) gave 2.78 (74%) of the N-benzyl derivative of 3 as a red syrup.

(3.) Hydrogenolysis of the N-benzyl derivative of 3

The benzyl protected pyridone 4 (1.33 g, 4.48 mmol) was stirred withPd(OH)₂ (20 wt. %) in acetic acid under a H₂ -filled balloon at roomtemperature overnight. The solution was filtered and the acetic acidsolvent was removed by rotary evaporation. Toluene was added to theresidue, and the solution was again evaporated to remove the remainingacetic acid. The crude product (80%) was used directly in the followingO-methylation reaction.

Preparation of methoxypyridine 4 (n=1, R¹ =OCH₃)

The crude pyridone (80%×4.48 mmol) was stirred with a mixture of Ag₂ CO₃(2 mol equivalent), iodomethane (10 mol equivalent), and chloroform (50mL) in the dark at room temperature overnight. Filtration, concentrationand silica gel chromatography (40% ethyl acetate/hexanes as eluent) gave0.74 g (75% for the two steps) of product 5.

Preparation of β-Keto ester 5 (n=1, R¹ =OCH₃)

(1) The ketal 4(1.71 g) was refluxed in 5% HCl-acetone (1:1) overnight.Acetone was removed on a rotary evaporator and the aqueous layer wasbasified with solid NaHCO₃. The resulting mixture was extracted withethyl acetate. The organic extracts were washed with brine, dried overanhydrous MgSO₄, and filtered. Concentration and flash chromatography(30% ethyl acetate/hexanes) gave 1.16 g (85%) of the product as a stickysolid.

(2) The above ketone (1.16 g, 6.55 mmol) in 10 mL of dimethyl carbonatewas added dropwise to a mixture of KH (1.05 g, 26.2 mmol) in 40 mL ofdimethyl carbonate under nitrogen at room temperature. The mixture wasrefluxed for 3 h. The reaction was quenched with methanol, and thesolution was neutralized with a saturated NH₄ Cl solution. The methanolwas removed by rotary evaporation, and the aqueous residue was extractedwith ethyl acetate. The ethyl acetate extracts were washed with brine,dried over anhydrous MgSO₄, and filtered. Concentration and flashchromatography (20% ethyl acetate/hexanes) gave 1.34 g (87%) of 5 as asolid.

Preparation of Bridged Adduct 6 (n=1, R¹ =OCH₃, R² =R³ =CH₃)

The β-keto ester 5(502 mg, 2.15 mmol) was stirred with methacrolein(1.76 mL, 21.4 mmol) and 1,1,3,3-tetramethyl-guanidine (54 μL, 0.42mmol) in dry CH₂ Cl₂ at room temperature overnight. Concentration andflash chromatography (40% ethyl acetate/hexanes) gave 604 mg (93%) ofthe bridged adduct 6.

Dehydration of Alcohol 6

(1) Mesyl chloride (1.89 mL, 24.5 mmol) was added dropwise to a solutionof the alcohol 6(1.87 g, 6.13 mmol), triethylamine (8.46 mL, 61.3 mmol),and a catalytic amount of 4-N,N-dimethylaminopyridine in 50 mL of dryCH₂ Cl₂ at room temperature. The solution was stirred for 6 h at roomtemperature. The solution was diluted with CH₂ Cl₂, washed with NH₄ Cl(sat.), dried, and concentrated to give 2.26 g (96%) of the crudemesylate.

(2) The crude mesylate (2.26 g, 5.90 mmol) was heated with anhydrousNaOAc (0.48 g, 5.9 mmol) in AcOH at 120° C. under N₂ for 24 h. Theacetic acid was removed by rotary evaporation at 50° C. The residue wasdissolved in ethyl acetate, washed with saturated Na₂ CO₃ and brine, anddried. Evaporation of the ethyl acetate and flash chromatography of theresidue (20% and then 40% ethyl acetate/hexanes) gave 521 mg (31% or 47%based on 66% conversion) of 7 as a solid and 0.76 g (34%) of thestarting material.

Wittig Reaction of β-Keto Ester 7 (n=1,R¹ =OCH₃, R² =R³ =CH₃)

n-BuLi (2.57 mL, 3.80 mmol) was added dropwise to a mixture ofethyltriphenylphosphonium bromide (1.59 g, 4.28 mmol) in 15 mL of dryTHF at room temperature under nitrogen. The reaction mixture was stirredat room temperature for 30 min. and then cooled to 0° C. The ketone (273mg, 0.951 mmol) in 5 mL of dry THF was added dropwise to this mixture at0° C. The resulting mixture was allowed to warm to room temperature, andstirred at room temperature for 4 h. The reaction was quenched withwater. The THF was removed by rotary evaporation, and the aqueousresidue was extracted with ethyl acetate. The ethyl acetate extractswere washed with brine, dried, and concentrated. Flash chromatography(10% ethyl acetate/hexanes) gave 208 mg (73%) of olefin 8 as a 10:90E/Z-mixture.

Isomerization of the Olefin Mixture 8(n=1,R¹ =OCH₃,R² =R³ =R⁴ =CH₃)

The olefin 8 (79 mg, 0.26 mmol) was heated with azoisobutyronitrile (87mg, 0.52 mmol) in 10 mL of thiophenol at 130° C. under nitrogen for 24h. The solution was cooled, diluted with CH₂ Cl₂, and washed with 10%NaOH (5 times) and brine. After drying and concentration, the crudeproduct was used directly in the next hydrolysis reaction. ¹ H NMRanalysis revealed olefin 9 to be comprised of an 80/20 mixture of the Eand Z-alkenes, respectively.

Preparation of Carbamate 10 (n=1, R¹ =OCH₃, R² =R³ =CH₃)

The crude ester (0.26 mmol, E/Z=80/20) was dissolved in a 1:1 mixture of20% NaOH and THF. Enough MeOH was added to convert the heterogeneousmixture to a homogenous one, and this solution was refluxed undernitrogen for 2 days. THF and MeOH were removed by rotary evaporation,and the aqueous residue was extracted with CH₂ Cl₂. These organicextracts were washed with brine, dried, and concentrated to give theunhydrolyzed Z-ester which can be recycled through the isomerizationstep. The 2 aqueous residue was adjusted to a pH of 7 with concentratedHCl. Extraction with CH₂ Cl₂, drying, and concentration gave the crudeacid which was further purified by column chromatography (20% ethylacetate/hexanes and then ethyl acetate) to afford 36 mg (61% based onthe E-ester) of pure acid.

Thionyl chloride (51μ, 0.65 mmol) was added dropwise to a solution ofthe acid (36 mg 0.13 mmol) in 5 mL of toluene under nitrogen at roomtemperature. The solution was heated at 80° C. for 2 h, and then cooledto room temperature. Sodium azide (82 mg, 1.3 mmol) was added, and themixture was heated at 80° C. overnight. The toluene was removed byrotary evaporation, 5 mL of MeOH was added, and the resulting mixturewas refluxed for 8 h. The methanol was removed by rotary evaporation,and the residue was dissolved in ethyl acetate. The solution was washedwith brine, dried, and concentrated. Flash chromatography (20% ethylacetate/hexanes) gave 15 mg (37%) of the urethane 10.

Huperzine A

Iodotrimethylsilane (50 μM, 0.35 mmol) was added dropwise to a solutionof the carbamate 10 (7 mg, 0.02 mmol) in 2 mL of chloroform undernitrogen at room temperature. The solution was then refluxed overnight.Methanol (2 mL) was added, and the solution was refluxed for anadditional 2 h. Concentration and flash chromatography on silica gelhalf-saturated with ammonia (3% methanol in chloroform) gave 4 mg (70%)of huperzine A along with 2 mg (30%) of the partially deprotectedcarbamate.

The following is the spectral data for EXAMPLE I:

2 (isomer ratio=85/15): R_(f) =0.30 (ethyl acetate); IR 2900-3700 (br.),3211, 3063, 2951, 1676, 1473, 1392, 1340, 1255, 1213, 1126, 1097, 1061,1020, 993, 947, 920, 733 cm⁻¹ ; ¹ H NMR δ 8.45 (br. s, 0.85 H), 7.73(br. s, 0.15 H), 4.83-4.87 (m, 0.85 H), 3.90-4.03 (m, 4 H), 1.51-2.56(four groups of multiplets, 9.15 H); mass spectrum (m/z) 209 (M⁺), 123,86, exact mass calcd. for C₁₁ H₁₅ NO₃ 209.1052, found 209.1051.

N-Benzyl derivative of 2: (isomer ratio=70/30): R_(f) =0.46 (ethylacetate); IR 2949, 2889, 1668, 1645, 1496, 1454, 1429, 1396, 1375, 1286,1192, 1145, 1101, 1061, 1026, 947, 698 cm⁻¹ ; ¹ H NMR δ 7.13-7.32 (m, 5H), 5.41 (d, 0.7 H, J=16.1 Hz), 4.84-4.87 (m. 1.3 H), 4.50 (d. 0.7 H,J=16.1 Hz), 3.91-4.03 (m, 4 H), 1.58-2.81 (four groups of multiplets,9.3 H); mass spectrum (m/z) 299 (M⁺), 213, 185, 91, exact mass calcd.for C₁₈ H₂₁ NO₃ 299.1521, found 299.1521.

N-Benzyl derivative of Pyridine 3: R_(f) =0.17 (ethyl acetate); IR 2957,2887, 1664, 1593, 1545, 1496, 1454, 1419, 1398, 1373, 1269, 1228, 1207,1167, 1113, 1062, 1028, 947, 862, 827, 733, 702 cm⁻¹ ; ¹ H NMR δ7.06-7.34 (m, 6 H), 6.57 (d, 1 H, J=9.3 Hz), 5.34 (s 2 H), 3.97-4.02 (m,4 H), 2.80 (t, 2 H, J=6.6 Hz), 2.73 (s, 2 H), 1.83 (t, 2 H, J=6.7 Hz);mass spectrum (m/z) 297 (M⁺), 206, 134, 91, exact mass calcd for C₁₈ H₁₉NO₃ 297.1365, found 297.1364.

3: mp=dec. above 250° C., IR 2930, 1639, 1620, 1554, 1506, 1464, 1446,1379, 1269, 1130, 1097, 1061, 1014, 949, 837, 696 cm⁻¹ ; ¹ H NMR δ 12.56(br. s, 1 H), 7.14 (d, 1 H, J=9.3 Hz), 6.40 (d, 1 H, J=9.3 Hz), 4.02 (s,4 H), 2.89 (t, 2 H, J=6.6 Hz), 2.71 (s, 2 H), 1.93 (t, 2 H, J=6.6 Hz),¹³ C NMR δ 165.0, 143.4, 141.8, 117.3, 111.9, 107.3, 64.6, 36.2, 30.1,25.7; mass spectrum (m/z) 207 (M⁺), 164, 134, 86, 69, 57, exact masscalcd for C₁₁ H₁₃ NO₃ 207.0895, found 207.0896.

4: mp =77.5°-78.5° C.; R_(f) =0.48 (40% ethyl acetate in hexanes); IR2942, 2885, 1601, 1581, 1478, 1466, 1457, 1429, 1420, 1313, 1259, 1120,1094, 1061, 1032, 1018, 947, 817 cm⁻¹ ; ¹ H NMR δ 7.22 (d, 1 H, J=8.3Hz), 6.52 (d, 1 H, J=8.3 Hz), 4.03 (s, 4 H), 3.88 (s, 3 H), 3.01 (t, 2H, J=6.8 Hz), 2.89 (s, 2 H), 2.01 (t, 2 H, J=6.8 Hz); mass spectrum(m/z) 221 (M⁺), 148, 134, 64, exact mass calcd for C₁₂ H₁₅ NO₃ 221.1052,found 221.1053.

Ketone derived from 4: R_(f) =0.44 (40% ethyl acetate in hexanes); IR2945, 2916, 2891, 1712, 1604, 1582, 1482, 1430, 1337, 1318, 1309, 1296,1267, 1195, 1188, 1182, 1166, 1108, 1032, 859, 825 cm⁻¹ ; ¹ H NMR δ 7.30(d, 1 H, J=8.3 Hz), 6.61 (d, 1 H, J=8.3 Hz), 3.93 (s, 3 H), 3.51 (s, 2H), 3.16 (t, 2 H, J=6.9 Hz), 2.66 (t, 2 H, J=6.9 Hz); ¹³ C NMR δ 209.4,162.7, 153.5, 138.8, 120.2, 108.8, 53.4, 42.5, 38.0, 30.9; mass spectrum(m/z) 177 (M⁺), 162, 148, 106, exact mass calcd for C₁₀ H₁₁ NO₂177.0790, found 177.0790.

5: R_(f) =0.33 (20% ethyl acetate in hexanes); IR 2954, 2895, 2837,1641, 1603, 1568, 1477, 1448, 1427, 1317, 1263, 1226, 1116, 1059, 1035,1016, 941, 918, 825, 785, 640, 625 cm⁻¹ ; ¹ H NMR δ 13.16 (s, 1 H), 7.90(d, 1 H, J=8.7 Hz), 6.56 (d, 1 H, J=8.7 Hz), 3.91 (s, 3 H), 3.90 (s, 3H), 2.94 (t, 2 H, J=8.7 Hz), 6.56 (d, 1 H, J=8.7 Hz), 3.91 (s, 3H), 3.90(s, 3 H), 2.94 (t, 2 H, J=7.8 Hz), 6.56 (d, 1 H, J=8.7 Hz), 3.91 (s, 3H), 3.90 (s, 3 H), 2.94 (t, 2 H, J=7.8 Hz), 2.63 (t, 2 H, J=7.8 Hz); ¹³C NMR δ 176.7, 171.9, 161.1, 151.1, 136.1, 119.8, 107.2, 98.2, 53.3,51.7, 29.9, 29.0; mass spectrum (m/z) 235 (M⁺), 203, 148, exact masscalcd for C₁₂ H₁₃ NO₄ 235.0845, found 235.0845.

6: R_(f) =0.30-0.35 (40% ethyl acetate in hexanes); IR 3100-3600 (br),2953, 1743, 1603, 1576, 1481, 1423, 1325, 1269, 1155, 1118, 1078, 1034,983, 827, 758 cm⁻¹ ; ¹ H NMR (one of the isomers) δ 7.02 (d, 1 H, J=8.6Hz), 6.60 (d, 1 H, J=8.6 Hz), 3.91 (s, 3 H), 3.81 (s, 3 H), 3.62-3.69(m, 2 H), 3.03-3.25 (m, 2 H), 2.23 (br. s, --OH), 1.98-2.04 (m, 2 H),1.48-1.59 (m, 1 H), 1.03 (d, 3 H, J=6.4 Hz), mass spectrum (m/z) 305(M⁺), 273, 248, 188, 55, exact mass calcd for C₁₆ H₁₉ NO₅ 305.1263,found 305.1264.

7: R_(f) =0.27 (20% ethyl acetate in hexanes); IR 2947, 1745, 1603,1576, 1479, 1423, 1327, 1263, 1194, 1138, 1111, 1082, 1024, 831 cm⁻¹ ; ¹H NMR (500 MHz) δ 7.11 (d, 1 H, J=8.6 Hz), 6.62 (d, 1 H, J=8.6 Hz),5.42-5.43 (m, 1 H), 3.92 (s, 3 H), 3.76 (s, 3 H), 3.36-3.42 (m, 2 H),3.18 (d, 1 H, J=18.2 Hz), 3.15 (m, 1 H), 2.13 (d, 1 H, J=17.5 Hz) 1.60(s, 3 H), ¹³ C NMR δ 207.5, 171.4, 163.2, 150.7, 137.7, 133.6, 126.4,123.8, 109.6, 60.1, 53.4, 52.7, 46.9, 46.0, 40.4, 22.3; mass spectrum(m/z) 287 (M⁺), 255, 228, 200, 184, exact mass calcd for C₁₆ H₁₇ NO₄287.1158, found 287.1157.

8 (Z-olefin); R_(f) =0.39 (20% ethyl acetate in hexanes); IR 2909, 1732,1601, 1578, 1558, 1475, 1423, 1321, 1252, 1205, 1151, 1111, 1086, 1030,1003, 902, 827, 735, 638 cm⁻¹ ; ¹ H NMR δ 7.09 (d, 1 H, J=8.5 Hz), 6.54(d, 1 H, J=8.6 Hz), 5.51 (q, 1 H, J=7.3 Hz), 5.40-5.42 (m, 1 H), 3.89(s, 3 H), 3.71 (s, 3 H), 2.99-3.19 (m, 3 H), 2.81 (d, 1 H. J=16.5 Hz);2.21 (d, 1 H, J=17.0 Hz), 1.57 (S, 3 H), 1.51 (d, 1 H, J=16.5 Hz); 2.21(d, 1 Ho J=17.0 Hz), 1.57 (s, 3 H), 1.51 (d, 3 H, J=7.3 Hz); massspectrum (m/z) 299 (M⁺), 240, 57, exact mass calcd for C₁₈ H₂₁ NO₃299.1521, found 299.1521.

Acid from 9: R_(f) =0.39 (ethyl acetate); IR 2500-3500 (br), 2932, 2594,1705, 1599, 1578, 1477, 1423, 1379, 1323, 1269, 1128, 1111, 1076, 1030,956, 908, 823, 777, 760, 735, 681, 646 cm⁻¹ ; ¹ H NMR δ 7.25 (d, 1 H,J=8.5 Hz), 6.57 (d, 1 H. J=8.5 Hz), 5.40-5.42 (m, 1 H), 5.31 (q, 1 H,J=6.7 Hz), 3.89 3.89 (s, 3 H), 3.62 (m, 1 H), 2.84-3.12 (m, 3 H), 2.18(d, 1 H, J=17.0 Hz); 2.74 (d, 3 H, J=6.8 Hz), 1.54 (s, 3 H), 2.18 (d, 1H, J=17.0 Hz); 2.74 (d, 3 H, J=6.8 Hz), 1.54 (s, 3 H); mass spectrum(m/z) 285 (M⁺), 240, 84, exact mass calcd for C₁₇ H₁₉ NO₃ 285.1365,found 285.1365.

10: R_(f) =0.15 (20% ethyl acetate in hexanes); IR 3331 (br), 2930,1716, 1597, 1581, 1558, 1522, 1475, 1421, 1321, 1304, 1257, 1103, 1068,1032, 914, 827, 777, 733 cm⁻¹ ; ¹ H NMR δ 7.56 (d, 1 H, J=8.6 Hz), 6.55(d, 1 H, J=8.6 Hz), 5.54-5.56 (m, 1 H), 5.36 (q, 1 H, J=6.8 Hz), 4.98(s, --NH), 3.88 (s, 3 H), 3.66 (br.s, 1 H), 3.62 (s, 3 H) 3.07 (br. d, 1H. J=17.4 Hz), 2.82 (dd, 1 H, J=16.7, 1.6 Hz), 2.57 (br. d, 1 H, J=15Hz), 2.23 (d, 1 H, J=15.6 Hz), 1.72 (d, 3 H, J=6.8 Hz), 1.51 (s, 3 H);mass spectrum 314 (M⁺), 224, 84, 69, exact mass calcd for C₁₈ H₂₂ N₂ O₃314.1630, found 314.1630.

Synthetic Huperzine-A: R_(f) =0.10 (basic SiO₂ CHCl₃ -Acetone-MeOH:50/45/5); IR 3277, 2928, 1655, 1616, 1558, 1458, 1406, 1377, 1306, 1174,1118, 912, 833, 769, 731, 659 cm⁻¹ ; ¹ H NMR δ 12.42 (br. s, pyridone--NH), 7.90 (d, 1 H, J=9.3 Hz), 6.42 (d, 1 H, J=9.6 Hz), 5.49 (q, 1 H,J=6.7 Hz), 5.42 (m, 1 H), 3.61 (m, 1 H), 2.89 (dd, 1 H, J=16.8, 5.1 Hz),2.70 (d, 1 H, J=15.9 Hz), 2.14 (br. s, 2 H), 1.68 (d, 3 H. J=6.6 Hz),1.61 (br. s, --NH₂), 1.55 (s, 3 H); mass spectrum (m/z) 242 (M⁺), 227,187, 57, exact mass calcd for C₁₅ H₁₈ N₂ O 242.1419, found 242.1419.

6.2 Example II

Biological Activity of Huperzine A and the 1-Carbon Analog of HuperzineA

The ability of natural huperzine A and synthetic racemic huperzine A,and the propylidene compound (general formula IV, R₄ ³ =R₄ ⁴ =CH₃, R₄ ⁵=R₄ ⁶ =R₄ ⁷ =H, n=1, p=O, with a double bond between carbon 8 and carbon15), which is the 1-carbon analog of huperzine A, to inhibit thecholinesterase enzymes was determined.

Method

Rats were killed by decapitation and the brains were rapidly extirpated.The cortex was dissected out on ice according to the method of Glowinskiand Iversen. (See J. Neurochem. 13, 655 (1966)). Samples werehomogenized in ice cold 0.32M sucrose. Homogenates were centrifuged at1000×g for 10 minutes to remove cell nuclei and heavy debris. Thesupernatant was then aspirated off and spun again (12000×g) for 20minutes to form a pellet (Whittaker's P₂ fraction) containingsynaptosomes and mitochondria. See E. G. Gray et al., J. Anatomy,96,70(1962). The pellet was resuspended in 0.32M sucrose. A portion ofthis synaptosome-rich fraction was added in triplicate to ice-cold pH7.4 Krebs-Ringer medium.

Assay of acetylcholinesterase was carried out according to the method ofJohnson and Russell. See C. D. Johnson et al., Anal. Biochem.,64,229(1978). Acetylcholine labelled in the acetate moiety wasenzymatically hydrolyzed by incubation for 10 minutes at roomtemperature in the presence of the above synaptosome-rich fractioncontaining endogenous acetylcholinesterase enzyme. The reaction wasterminated by addition of a "stopping mixture" containing chloroaceticacid (1.0M), sodium hydroxide (0.5M) and sodium chloride (2.0M) to thereaction vial. Toluene-based scintillation fluid was added to the vial,to extract the released labelled acetate into the organic phase. Underthese conditions the unhydrolyzed labelled acetylcholine remainsunextracted in the small aqueous reaction volume from which its weakbeta-particles of decay do not escape to excite the scintillator. Thus,the sample can be counted directly in the same reaction vial, in whichthe hydrolysis of sample by acetylcholinesterase has occurred.

Inhibition of cholinesterase activity was estimated, in triplicate, inthe presence of a wide range of concentrations (10⁻⁹ to 10⁻³ M).

Results

The results are shown in Table I.

                  TABLE I    ______________________________________    EXTENT OF CHOLINESTERASE ENZYME INHIBITION    Structural Name                 IC.sub.50 /IC.sub.50 of    Synthesized  M.W.      IC.sub.50                                    Huperzine A*    ______________________________________    Natural Huperzine A                 242       10.sup.-7 M                                    --    Synthesized Huperzine A                 242       6 × 10.sup.-7 M                                    1.0    1-carbon Analog                 256       10.sup.-4 M                                    166.6    ______________________________________     *The smaller this number, the more potent the compound.

As shown in Table 1, synthetic racemic huperzine A had an IC₅₀ of 6×10⁻⁷M. This was very similar to the IC₅₀ value of natural huperzine A (10⁻⁷M). Also, the 1-carbon analog inhibited the cholinesterase enzyme, butto a lesser extent than huperzine A.

What is claimed is:
 1. A compound having the general formula II: ##STR12## wherein: R₁ ¹ is selected from the group consisting of H and C₁ -C₈ linear or branched alkoxy;R₁ ² is C₁ -C₈ linear or branched alkyl; and n₁ is an integer from 0 to
 4. 2. The compound according to claim 1 wherein R₁ ¹ is a C₁ -C₈ linear or branched alkoxy.
 3. The compound according to claim 2 wherein:R₁ ¹ is OCH₃ ; R₁ ² is CH₃ ; and n₁ is
 1. 